Lck (IC50 = 249 nM); PAK5 (IC50 = 166 nM)

PRT062607 (P505-15) is a brand-new, extremely effective, and strong small-molecule inhibitor of Syk that is taken orally. Two distinct pure kinase assays are used to investigate PRT062607's efficacy against its target kinase Syk. Half-maximum Syk inhibition needed 6±0.2 nM (mean±SEM) according to a FRET test. A radioactive enzyme assay yields a Syk IC50 of 2.1±0.4 nM (mean±SEM), indicating similar potency. PRT062607 effectively suppresses Fcε receptor 1-mediated basophil degranulation (IC50 0.15 μM) and B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 μM, respectively) in human whole blood[1].

When PRT062607 (P505-15) is administered orally to mice in the CAIA model, the average reduction in paw inflammation, as determined by daily scoring of inflammation in comparison to vehicle controls, is 12, 44, and 87%. At the conclusion of the study, the average plasma concentration (C average over 24 hours) was determined to be 0.38, 0.95, and 1.47 μM. Mice given 30 mg/kg of PRT062607 showed a considerable reduction in joint degeneration, to the point that the mice could not be distinguished from normal mice. By the end of the research, the majority of the animals (seven out of eight) in the rat CIA model (mean inflammation score±SEM=0.63±1.1; p<0.0001 versus vehicle) had totally suppressed inflammation due to the high dose of PRT062607 (15 mg/kg bid)[1].

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In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 μM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 μM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 μM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.[2]

SYK Autophosphorylation. [1]
Ramos cells (107 cells per experiment) were preincubated for 30 minutes with or without PRT062607 (P505-15)  and then stimulated (30 minutes at 37°C) with 1 μg/ml anti-IgM. Signaling was terminated by resuspending cells in RIPA lysis buffer (50 mM Tris 7.4, 1% NP40, 0.5% sodium deoxycholate, 150 mM NaCl, 0.5 mM EDTA) containing fresh protease and phosphatase inhibitors and incubated on ice for 1 hour. Following centrifugation, protein lysates were pre-cleared with protein A/G sepharose beads. Lysates were incubated overnight (4°C) with rabbit anti-SYK antibody and precipitated with protein A/G sepharose beads. Washed beads were denatured and proteins were resolved by 10% SDS-PAGE, immunoblotted with rabbit anti-SYK antibody, stripped, and reblotted with rabbit anti-pSYK Y526/526 antibody.

Intracellular Phospho-flow Cytometry. [1]
Ramos cells (0.5 × 106) were suspended in 200 μl fresh media (RPMI plus 10% fetal calf serum) and pretreated with vehicle or PRT062607 (P505-15)  (30 minutes at 37°C). Cells were left unstimulated or were stimulated with 1 μg/ml goat F(ab)’2 anti-IgM (Life Technologies) for 10 minutes. Ficoll purified (2 × 106) frozen viable CLL cells obtained from peripheral blood of CLL patients (n = 7) were thawed at 37°C, washed in 10 ml RPMI media plus 10% fetal calf serum by centrifugation, and resuspended at 106 cells/ml in the same media. Aliquots (200 μl) of cells were treated with vehicle or PRT062607 (P505-15)  for 30 minutes prior to stimulation with 0.3 mM H2O2 (Reth 2002; Irish et al., 2006) (8.8 M stock) followed by addition of 6 μg of a 1:1 mixture of goat F(ab’)2 anti-human IgG and anti-human IgM for 10 minutes. Signaling was stopped by addition of 60 μl of a 16% paraformaldehyde solution followed by 10-minute incubation at room temperature. Fixed cells were washed twice in ice-cold phosphate buffered saline (PBS) by centrifugation, suspended in a 50% methanol solution in PBS prechilled to −20°C, and stored at 4°C overnight. Cells were stained for intracellular phosphoflow cytometry by washing the permeabilized cells twice in PBS containing 1% BSA, followed by incubation in the same buffer containing various antibodies, as indicated in the results, per manufacturer instructions. Cells were washed once more in PBS containing 1% BSA prior to FACS analysis, in which at least 2,000 events were collected using a BD Biosciences FACS Calibur. Data were analyzed using FlowJo software.

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Apoptosis. [1]
Apoptosis was measured using the PE-conjugated monoclonal active caspase-3 antibody apoptosis kit. Cells were suspended in growth media (0.5 × 106 cells/ml) and treated with the indicated concentrations of PRT062607 (P505-15)  or vehicle control for 72 hours prior to FACS analysis. In some experiments, SU-DHL6 (0.5 × 106 cells) was mixed with 100 µl heparinized human whole blood. Samples were treated with 1 µM PRT062607 (P505-15)  for 24 hours and then surface stained with anti-human CD19 antibody prior to preparation for FACS analysis of active caspase-3.

In Vitro and In Vivo Stimulation with Anti-IgD. [1]
Spleens were harvested from Balb/c mice and separated into a single cell suspension using a single cell strainer. Cells were collected in PBS containing 1% BSA by centrifugation, washed once in the same buffer, and resuspended in RPMI (containing 10% FCS) at 106 cells/ml. Aliquots (190 µl) were then treated with various concentrations of PRT062607 (P505-15)  for 1 hour prior to stimulating overnight in a 37°C tissue culture incubator with 1 µl anti-mouse immunoglobulin D (IgD). Following stimulation, cells were stained with anti-CD80/86 FITC and anti-CD45R/B220 APC for 30 minutes at room temperature, washed once in PBS containing 1% BSA, and resuspend in 300 μl of the same buffer for collection of 2000 CD45-positive events by flow cytometry. Balb/c mice (n = 5 per group) received daily oral BID doses of vehicle (0.5% methylcellulose in water) or PRT062607 (P505-15)  (15 mg/kg) for a total of 5 days. On study day 1, 1 hour after the first oral dose, mice received a single 200-μl subcutaneous injection of control goat serum or anti-IgD serum. On study day 5, mice were anesthetized with SC ketamine cocktail and exsanguinated via cardiac puncture. Spleens were weighed and then sectioned and stained with hematoxylin and eosin for histology.

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Xenograft Studies. [1]
NOD/SCID mice were acclimated in-house at least 3 days prior to use. Ramos cells (3 × 106) were injected subcutaneously into the hind flank area of conscious mice using a 27-gauge needle in an injection volume of less than 0.5 ml. Following injection, mice were randomized into treatment groups (n = 15) and dosed twice daily by oral gavage with vehicle (0.5% methylcellulose in water) or 10, 15, or 20 mg/kg PRT062607 (P505-15) . Body weights were obtained at least once per week, and caliper measurements of tumors were determined twice per week beginning when palpable tumors were formed until the end of the study. Tumor volume was assessed by caliper measurement using a formula [maximum length × width × height × pi/6]. Twice daily dosing of vehicle or PRT062607 (P505-15)  continued until the vehicle or any treatment group exhibited tumors that exceeded 1.5 g in size. At the time of termination (5 weeks post-Ramos inoculation) the mice were anesthetized with a ketamine cocktail. A blood sample via cardiac puncture was obtained for CBC and plasma concentration determination, and then the mice were euthanized via cervical dislocation. Tumors were excised, weighed, and then snap frozen in liquid nitrogen for determination of concentration of PRT062607 (P505-15)  in the tumor tissue. Statistical comparison of tumor weights across groups was performed using a t-test.

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10, 15, or 20 mg/kg; Oral administration

NOD/SCID mice injected with Ramos cells

[1]. Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis. J Pharmacol Exp Ther. 2012 Feb;340(2):350-9.

[2]. The selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and function in vitro and in vivo and augments the activity of fludarabine in chronic lymphocytic leukemia. J Pharmacol Exp Ther. 2013 Feb;344(2):378-87.

[3]. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia. 2012 Jul;26(7):1576-83.

Given its preclinical activity and specificity, especially when compared with previously studied SYK inhibitors, P505-15 is an attractive compound for clinical development. Additional studies aimed at defining the biologic characteristics associated with SYK sensitivity are needed. Exploring additional P505-15 combinations including chemotherapy, monoclonal antibodies, or novel combinations of kinase inhibitors targeting multiple branches of signaling pathways, may identify further therapeutic opportunities. A dose finding study using P505-15 in healthy volunteers has been completed and includes single and multiple dosing regimens.[1]

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B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.[2]

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Syk is a protein tyrosine kinase that couples B-cell receptor (BCR) activation with downstream signaling pathways, affecting cell survival and proliferation. Moreover, Syk is involved in BCR-independent functions, such as B-cell migration and adhesion. In chronic lymphocytic leukemia (CLL), Syk becomes activated by external signals from the tissue microenvironment, and was targeted in a first clinical trial with R788 (fostamatinib), a relatively nonspecific Syk inhibitor. Here, we characterize the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, in assays that model CLL interactions with the microenvironment. PRT318 and P505-15 effectively antagonize CLL cell survival after BCR triggering and in nurse-like cell-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration toward the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and extracellular signal-regulated kinase phosphorylation after BCR triggering. These findings demonstrate that the selective Syk inhibitors PRT318 and P505-15 are highly effective for inhibition of CLL survival and tissue homing circuits, and support the therapeutic development of these agents in patients with CLL, other B-cell malignancies and autoimmune disorders.[3]

C19H23N9O.C2H4O2

453.49758

393.202

1370261-96-3

PRT062607 Hydrochloride;1370261-97-4

44462758

White to off-white solid powder

1.5±0.1 g/cm3

707.6±70.0 °C at 760 mmHg

381.7±35.7 °C

0.0±2.3 mmHg at 25°C

1.783

0.74

4

8

6

29

544

2

NC(C1=CN=C(N[C@H]2[C@@H](N)CCCC2)N=C1NC3=CC=CC(N4N=CC=N4)=C3)=O

TXGKRVFSSHPBAJ-JKSUJKDBSA-N

InChI=1S/C19H23N9O/c20-15-6-1-2-7-16(15)26-19-22-11-14(17(21)29)18(27-19)25-12-4-3-5-13(10-12)28-23-8-9-24-28/h3-5,8-11,15-16H,1-2,6-7,20H2,(H2,21,29)(H2,22,25,26,27)/t15-,16+/m0/s1

2-[[(1R,2S)-2-aminocyclohexyl]amino]-4-[3-(triazol-2-yl)anilino]pyrimidine-5-carboxamide

PRT062607; 1370261-96-3; PRT-062607; 2-[[(1R,2S)-2-aminocyclohexyl]amino]-4-[3-(triazol-2-yl)anilino]pyrimidine-5-carboxamide; K9C42672RH; P505-15; BIIB057; UNII-K9C42672RH;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)